iBRIDGE: A Data Integration Method to Identify Inflamed Tumors from Single-cell RNA-Seq Data and Differentiate Cell Type-Specific Markers of Immune-Cell Infiltration.

The development of immune checkpoint-based immunotherapies has been a major advancement in the treatment of cancer, with a subset of patients exhibiting durable clinical responses. A predictive biomarker for immunotherapy response is the preexisting T-cell infiltration in the tumor immune microenvironment (TIME). Bulk transcriptomics-based approaches can quantify the degree of T-cell infiltration using deconvolution methods and identify additional markers of inflamed/cold cancers at the bulk level. However, bulk techniques are unable to identify biomarkers of individual cell types. Although single-cell RNA sequencing (scRNA-seq) assays are now being used to profile the TIME, to our knowledge there is no method of identifying patients with a T-cell inflamed TIME from scRNA-seq data. Here, we describe a method, iBRIDGE, which integrates reference bulk RNA-seq data with the malignant subset of scRNA-seq datasets to identify patients with a T-cell inflamed TIME. Using two datasets with matched bulk data, we show iBRIDGE results correlated highly with bulk assessments (0.85 and 0.9 correlation coefficients). Using iBRIDGE, we identified markers of inflamed phenotypes in malignant cells, myeloid cells, and fibroblasts, establishing type I and type II interferon pathways as dominant signals, especially in malignant and myeloid cells, and finding the TGFß-driven mesenchymal phenotype not only in fibroblasts but also in malignant cells. Besides relative classification, per-patient average iBRIDGE scores and independent RNAScope quantifications were used for threshold-based absolute classification. Moreover, iBRIDGE can be applied to in vitro grown cancer cell lines and can identify the cell lines that are adapted from inflamed/cold patient tumors.

Resveratrol Ameliorates Lipopolysaccharide-Induced Sudden Sensorineural Hearing Loss in In Vitro Model through Multitarget Antiapoptotic Mechanism Based on Network Pharmacology and Molecular Docking.

Objective: To explore the effects of resveratrol (RSV) on hair cell apoptosis caused by sudden sensorineural hearing loss (SSNHL) and its effect on lipopolysaccharide-induced apoptosis of HEI-OC1 cells. Methods: We used the network pharmacology method to screen molecules related to RSV for the treatment of SSNHL and analyzed these molecules and their enriched biological processes and signaling pathways through Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. We selected hub genes related to apoptosis using protein-protein interaction (PPI) analysis for in vitro and molecular docking verification. Results: Eighty overlapping genes were identified as potential targets for RSV treatment of SSNHL. Further GO analysis showed that the biological processes were mainly related to toxicity, cell proliferation, and lipopolysaccharide reactions. KEGG analysis showed that the AGE-RAGE signaling pathway in diabetic complications, Kaposi's sarcoma-associated herpesvirus infection, FoxO signaling pathway, PI3K-Akt signaling pathway, and other inflammatory signaling pathways were concentrated. AKT1, STAT3, JUN, TNF, TP53, MAPK3, CASP3, and VEGFA were screened as HUB genes using PPI analysis. The apoptosis-related proteins TNF, CASP3, AKT1, and TP53 were selected for in vitro experiments, which showed that mRNA was significantly different before and after RSV intervention, confirming that the corresponding protein receptors could bind well with RSV. Conclusion: RSV mainly affects the prognosis of SSNHL through anti-inflammatory effects and may improve hair cell apoptosis caused by inflammatory factors through multitargeted interventions involving TNF, CASP3, AKT1, and TP53.

VISTA is an activating receptor in human monocytes.

As indicated by its name, V-domain Ig suppressor of T cell activation (VISTA) is thought to serve primarily as an inhibitory protein that limits immune responses. VISTA antibodies can dampen the effects of several concomitantly elicited activation signals, including TCR and TLR activation, but it is currently unclear if VISTA agonism could singly affect immune cell biology. In this study, we discovered two novel VISTA antibodies and characterized their effects on human peripheral blood mononuclear cells by scRNA/CITE-seq. Both antibodies appeared to agonize VISTA in an Fc-functional manner to elicit transcriptional and functional changes in monocytes consistent with activation. We also used pentameric VISTA to identify Syndecan-2 and several heparan sulfate proteoglycan synthesis genes as novel regulators of VISTA interactions with monocytic cells, adding further evidence of bidirectional signaling. Together, our study highlights several novel aspects of VISTA biology that have yet to be uncovered in myeloid cells and serves as a foundation for future research.

Phase I study of ABBV-428, a mesothelin-CD40 bispecific, in patients with advanced solid tumors.

BACKGROUND: CD40 agonist immunotherapy can potentially license antigen-presenting cells to promote antitumor T-cell activation and re-educate macrophages to destroy tumor stroma. Systemic administration of CD40 agonists has historically been associated with considerable toxicity, providing the rationale for development of tumor-targeted immunomodulators to improve clinical safety and efficacy. This phase I study assessed the safety, tolerability, preliminary antitumor activity, and preliminary biomarkers of ABBV-428, a first-in-class, mesothelin-targeted, bispecific antibody designed for tumor microenvironment-dependent CD40 activation with limited systemic toxicity. METHODS: ABBV-428 was administered intravenously every 2 weeks to patients with advanced solid tumors. An accelerated titration (starting at a 0.01 mg/kg dose) and a 3+3 dose escalation scheme were used, followed by recommended phase II dose cohort expansions in ovarian cancer and mesothelioma, tumor types associated with high mesothelin expression. RESULTS: Fifty-nine patients were treated at doses between 0.01 and 3.6 mg/kg. The maximum tolerated dose was not reached, and 3.6 mg/kg was selected as the recommended phase II dose. Seven patients (12%) reported infusion-related reactions. Treatment-related grade ≥3 treatment-emergent adverse events were pericardial effusion, colitis, infusion-related reaction, and pleural effusion (n=1 each, 2%), with no cytokine release syndrome reported. The pharmacokinetic profile demonstrated roughly dose-proportional increases in exposure from 0.4 to 3.6 mg/kg. Best response was stable disease in 9/25 patients (36%) treated at the recommended phase II dose. CD40 receptor occupancy >90% was observed on peripheral B-cells starting from 0.8 mg/kg; however, no consistent changes from baseline in intratumoral CD8+ T-cells, programmed death ligand-1 (PD-L1+) cells, or immune-related gene expression were detected post-ABBV-428 treatment (cycle 2, day 1). Mesothelin membrane staining showed greater correlation with progression-free survival in ovarian cancer and mesothelioma than in the broader dose escalation population. CONCLUSIONS: ABBV-428 monotherapy exhibited dose-proportional pharmacokinetics and an acceptable safety profile, particularly for toxicities characteristic of CD40 agonism, illustrating that utilization of a tumor-targeted, bispecific antibody can improve the safety of CD40 agonism as a therapeutic approach. ABBV-428 monotherapy had minimal clinical activity in dose escalation and in a small expansion cohort of patients with advanced mesothelioma or ovarian cancer. TRIAL REGISTRATION NUMBER: NCT02955251.

A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity.

Agonistic CD40 monoclonal antibodies (mAb) have demonstrated some clinical activity, but with dose-limiting toxicity. To reduce systemic toxicity, we developed a bispecific molecule that was maximally active in the presence of a tumor antigen and had limited activity in the absence of the tumor antigen. LB-1 is a bispecific molecule containing single-chain Fv domains targeting mouse CD40 and the tumor antigen mesothelin. LB-1 exhibited enhanced activity upon binding to cell-surface mesothelin but was less potent in the absence of mesothelin binding. In a mouse model implanted with syngeneic 4T1 tumors expressing cell-surface mesothelin, LB-1 demonstrated comparable antitumor activity as an agonistic CD40 mAb but did not cause elevation of serum cytokines and liver enzymes, as was observed in anti-CD40-treated mice. The results from our study of LB-1 were used to develop a human cross-reactive bispecific molecule (ABBV-428) that targeted human CD40 and mesothelin. ABBV-428 demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of cultured or implanted PC3 tumor cell growth after immune activation. Although expression of cell-surface mesothelin is necessary, the bispecific molecules induced immune-mediated antitumor activity against both mesothelin+ and mesothelin- tumor cells. ABBV-428 represents a class of bispecific molecules with conditional activity dependent on the binding of a tumor-specific antigen, and such activity could potentially maximize antitumor potency while limiting systemic toxicity in clinical studies.

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells.

Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis.

INTRODUCTION: Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20- plasmablasts is noted. The strong expression of CD319 on CD20- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target. METHODS: PDL241, a novel humanized IgG1 monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys. RESULTS: PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319+ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG1 and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed. CONCLUSIONS: The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.

Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb.

BACKGROUND: The receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer. METHODS: Expression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR. RESULTS: Overexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis. CONCLUSIONS: TweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.

Apolipoprotein (apo) E4 enhances amyloid beta peptide production in cultured neuronal cells: apoE structure as a potential therapeutic target.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid beta (Abeta) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Abeta production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Abeta production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in Abeta production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and Abeta production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.

Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy.

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20(+) mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.

Identification of cathepsin B as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach.

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated Abeta42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying Abeta-induced microglial activation, we analyzed the expression levels of transcripts isolated from Abeta42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by Abeta42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in Abeta-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by Abeta42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by Abeta-activated inflammatory response.

[The adjustable, folding and multi-function mount].

An adjustable folding mount is presented in this paper. It is a multi-functional device for one or more patient's infusions, continuous traction and functional training, and life nursing etc.